Summary/Abstract The overall objective of this project is to test and prioritize aminospectinomycin (amSPC) antibacterials for use as broad-spectrum agents against bacterial sexually transmitted diseases (STDs). The amSPCs, like their parent antibiotic, spectinomycin (SPC), act by inhibiting bacterial protein synthesis by binding to a ribosomal site that is unique among aminoglycosides and other protein synthesis inhibitors. Gonorrhea, caused by the bacterium Neisseria gonorrhoeae, is a STD that afflicts only humans. The disease can be asymptomatic and undiagnosed infections can lead to pelvic inflammatory disease and, ultimately, infertility or may disseminate, causing joint and skin manifestations. Therapeutic options consist of ceftriaxone (250 mg intramuscular in a single dose) plus azithromycin (1 gram orally in a single dose) or doxycycline dosed orally for 7 days. N. gonorrhoeae has acquired resistance to all agents that have been used as therapy, from the 1930s when sulfonamides were used as monotherapy until current times where resistance is developing to ceftriaxone and other extended-spectrum cephalosporins. Infections caused by Chlamydia trachomatis are even more prevalent than gonorrheal infections and, although antibiotic resistance is a smaller problem, people with chlamydial infections are often co-infected with other sexually transmitted bacterial pathogens such as Treponema palladum (syphilis), Haemophilus ducreyi and Mycoplasma genitalium. New small molecule drugs, especially those with synergy with new or existing antibiotics, rendering them useful for combination therapy, are desperately needed and the drug pipeline is very limited. We have discovered a series of novel amSPCs, which are derivatives of SPC, a second-line gonorrhea agent still in use outside of the United States. The structure-activity data available to date demonstrates an excellent (8-32-fold) improvement in in vitro potency compared to SPC and low cytotoxicity against multiple cell lines. The amSPCs represent a safe potential new treatment option for drug resistant gonorrhea and bacterial co-infections. The low oral bioavailability of the amSPCs suggests that they would be administered parenterally, as is standard for aminoglycoside anti- gonorrheal drugs and the current standard-of-care, cefriaxone. The goal of this Phase I research project is to develop the amSPCs as a parenteral therapy for the treatment of bacterial STDs by 1) maintaining potency against N. gonorrhoeae and C. trachomatis, 2) showing efficacy against other bacterial STDs and 3) demonstrating efficacy in a mouse model of gonorrhea. Our strategy is to evaluate and prioritize existing analogs (>100) to demonstrate a broad-spectrum potency against bacterial STD species, maintain low toxicity against mammalian cell lines and maintain or improve in vivo efficacy. In Phase II, we will evaluate the in vivo efficacy of the lead compounds emerging from Phase I in additional murine models of Ng infection and Ng-Ct co-infection and conduct pharmacokinetic and toxicology studies to establish an in vivo safety index and select the final broad-spectrum STD candidate suitable for IND-enabling preclinical GLP studies.